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Alternative lengthening of telomeres (ALT) is a telomerase‐independent telomere maintenance mechanism (TMM) with high prevalence in human osteosarcomas but remains unknown in canine osteosarcomas. The aim of this study was to evaluate the prevalence of ALT by detection of extra‐chromosomal circles of telomeric DNA and to assess clinical outcome in canine patients with spontaneous occurring appendicular osteosarcoma. Fifty dogs with histopathological confirmed osteosarcomas were included into this study. Medical records were retrospectively analysed for patient characteristics, oncologic therapy and survival. DNA was isolated from archived FFPE tumour tissue specimens and applied for C‐ and G‐circle assay (CCA and GCA) and for telomeric content (TC) measurement with radiolabeled probes. ALT activity was detected for 10 of 50 (20%) cases by CCA. Four CCA positive cases were detected even with input DNA below 1 ng and demonstrated the high sensitivity of CCA for canine tumours. G‐circles and TC were not suitable to distinguish CCA positive and negative cases. CCA‐status showed an association with male gender and Rottweiler breed. Dogs with CCA positive osteosarcomas had shorter overall survival times than patients with CCA‐tumours and CCA‐status was a significant prognostic factor besides treatment in the Cox proportional hazard model. These findings make canine osteosarcomas an interesting model for comparative TMM research, but future studies are warranted to investigate if CCA‐status can serve as novel prognostic marker.

Background & Aims Recently, overexpression of the fibroblast growth factor receptor 3 (FGFR3) splice variants FGFR3‐IIIb and FGFR3‐IIIc was found in ~50% of hepatocellular carcinoma (HCC). Here, we aim to identify FGFR3‐IIIb/IIIc ligands, which drive the progression of HCC. Methods FACS, MTT assay and/or growth curves served to identify the FGFR3‐IIIb/IIIc ligand being most effective to induce growth of hepatoma/hepatocarcinoma cell lines, established from human HCC. The most potent FGF was characterized regarding the expression levels in epithelial and stromal cells of liver and HCC and impact on neoangiogenesis, clonogenicity and invasive growth of hepatoma/hepatocarcinoma cells. Results Among all FGFR3‐IIIb/IIIc ligands tested, FGF9 was the most potent growth factor for hepatoma/hepatocarcinoma cells. Replication and/or sprouting of blood/lymphendothelial cells was stimulated as well. FGF9 occurred mainly in stromal cells of unaltered liver but in epithelial cells of HCC. Every fifth HCC exhibited overexpressed FGF9 and frequent co‐upregulation of FGFR3‐IIIb/IIIc. In hepatoma/hepatocarcinoma cells FGF9 enhanced the capability for clonogenicity and disintegration of the blood and lymphatic endothelium, being most pronounced in cells overexpressing FGFR3‐IIIb or FGFR3‐IIIc, respectively. Any of the FGF9 effects in hepatoma/hepatocarcinoma cells was blocked completely by applying the FGFR1‐3‐specific tyrosine kinase inhibitor BGJ398 or siFGFR3, while siFGFR1/2/4 were mostly ineffective. Conclusions FGF9 acts via FGFR3‐IIIb/IIIc to enhance growth and aggressiveness of HCC cells. Accordingly, blockade of the FGF9‐FGFR3‐IIIb/IIIc axis may be an efficient therapeutic option for HCC patients.

Malignant cells ensure telomere maintenance by the alternative lengthening of telomeres (ALT) in the absence of telomerase activity (TA). The retrotransposons “long interspersed nuclear element-1” (LINE-1, L1) are expressed in malignant cells and are primarily known to contribute to complex karyotypes. Here we demonstrate that LINE-1 ribonucleoprotein particles (L1-RNPs) expression is significantly higher in ALT+- versus in TA+-human glioma. Analyzing a role of L1-RNP in ALT, we show that L1-RNPs bind to telomeric repeat containing RNA (TERRA), which is critical for telomere stabilization and which is overexpressed in ALT+ cells. In turn, L1-RNP knockdown (KD) abrogated the nuclear retention of TERRA, resulted in increased telomeric DNA damage, decreased cell growth and reduced expression of ALT characteristics such as c-circles and PML-bodies. L1-RNP KD also decreased the expression of Shelterin- and the ALT-regulating protein Topoisomerase IIIα (TopoIIIα) indicating a more general role of L1-RNPs in supporting telomeric integrity in ALT. Our findings suggest an impact of L1-RNP on telomere stability in ALT+ dependent tumor cells. As L1-RNPs are rarely expressed in normal adult human tissue those elements might serve as a novel target for tumor ablative therapy. Keywords: Telomere, TERRA, LINE-1, DNA damage response, Alternative lengthening of telomeres

The inadequate representation of enzymes which catalyze the activation/detoxification of xenobiotics in cells that are currently used in genotoxicity testing of chemicals leads to a high number of false positive results and the number of follow up studies with rodents could be reduced by use of more reliable in vitro models. We found earlier that several xenobiotic drug metabolizing enzymes are represented in the human derived liver cell line Huh6 and developed a protocol for micronucleus (MN) experiments which is in agreement with the current OECD guideline. This protocol was used to test 23 genotoxic and non-genotoxic reference chemicals; based on these results and of earlier findings (with 9 chemicals) we calculated the predictive value of the assay for the detection of genotoxic carcinogens. We found a sensitivity of 80% and a specificity of 94% for a total number of 32 chemicals; comparisons with results obtained with other in vitro assays show that the validity of MN tests with Huh6 is higher as that of other experimental models. These results are promising and indicate that the use of Huh6 cells in genetic toxicology may contribute to the reduction of the use of laboratory rodents; further experimental work to confirm this assumption is warranted.

Purpose Therapy response to neoadjuvant radiochemotherapy (nRCT) of locally advanced rectal cancer varies widely so that markers predicting response are urgently needed. Fibroblast growth factor (FGF) and FGF receptor (FGFR) signaling is involved in pro-survival signaling and thereby may result in radiation resistance. Methods In a cohort of 43 rectal cancer patients, who received nRCT, we analyzed protein levels of FGF 8 and its downstream target Survivin by immunohistochemistry to assess their impact on nRCT response. In vitro resistance models were created by exposing colorectal cancer cell lines to fractionated irradiation and selecting long-term survivors. Results Our findings revealed significantly higher FGF8 and Survivin staining scores in pre-treatment biopsies as well as in surgical specimens of non-responsive compared to responsive patients. Functional studies demonstrated dose-dependent induction of FGF8 mRNA expression in mismatch-incompetent DLD1 cells already after one dose of irradiation. Surviving clones after one or two series of radiation were more resistant to an additional radiation fraction than non-irradiated controls and showed a significant increase in expression of the FGF8 receptor FGFR3 and of Survivin on both the RNA and the protein levels. Conclusion The results of this study suggest that FGF8 and Survivin contribute to radiation resistance in rectal cancer and may serve as markers to select patients who may not benefit from neoadjuvant radiotherapy. Electronic supplementary material The online version of this article (10.1007/s00432-018-2757-7) contains supplementary material, which is available to authorized users.

Epigenetic factors contribute to carcinogenesis, tumor promotion, and chemoresistance. Histone deacetylases (HDACs) are epigenetic regulators that primarily cause chromatin compaction, leading to inaccessibility of promoter regions and eventually gene silencing. Many cancer entities feature overexpression of HDACs. Currently, the role of HDACs in pancreatic neuroendocrine tumors (pNETs) is unclear. We analyzed the expression patterns of all HDAC classes (classes I, IIA, IIB, III, and IV) in 5 human tissue microarrays representing 57 pNETs resected between 1997 and 2013 and corresponding control tissue. All pNET cases were characterized clinically and pathologically according to recent staging guidelines. The investigated cases included 32 (56.1%) female and 25 (43.9%) male pNET patients (total n=57, 47.4% immunohistochemically endocrine positive). Immunohistochemical profiling revealed a significant up-regulation of all HDAC classes in pNET versus control, with different levels of intensity and extensity ranging from 1.5- to >7-fold up-regulation. In addition, expression of several HDACs (HDAC1, HDAC2, HDAC5, HDAC11, and Sirt1) was significantly increased in G3 tumors. Correlation analysis showed a significant association between the protein expression of HDAC classes I, III, and IV and rate of the pHH3/Ki-67-associated mitotic and proliferation index. Furthermore, especially HDAC5 proved as a negative predictor of disease-free and overall survival in pNET patients. Overall, we demonstrate that specific members of all 4 HDAC classes are heterogeneously expressed in pNET. Moreover, expression of HDACs was associated with tumor grading, proliferation markers, and patient survival, therefore representing interesting new targets in pNET treatment.

// Mohamed A. Ahmed 1, 2 , Edgar Selzer 3 , Wolfgang Dorr 3, 4 , Gerd Jomrich 5 , Felix Harpain 5 , Gerd R. Silberhumer 5 , Leonhard Mullauer 6 , Klaus Holzmann 1 , Bettina Grasl-Kraupp 1 , Michael Grusch 1 , Walter Berger 1 , Brigitte Marian 1 1 Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, Austria 2 Radiation Biology Department, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, Egypt 3 Department of Radiotherapy and Radiobiology, Medical University of Vienna, Austria 4 Christian Doppler Laboratory for Medical Radiation Research for Radiation Oncology, Medical University of Vienna, Austria 5 Department of Surgery, Medical University Vienna, Austria 6 Clinical Institute of Pathology, Medical University Vienna, Austria Correspondence to: Brigitte Marian, email: brigitte.marian@meduniwien.ac.at Keywords: FGFR4, colorectal cancer, radiotherapy, RAD51 Received: February 08, 2016 Accepted: September 12, 2016 Published: September 17, 2016 ABSTRACT In colorectal cancer (CRC), fibroblast growth factor receptor 4 (FGFR4) is upregulated and acts as an oncogene. This study investigated the impact of this receptor on the response to neoadjuvant radiotherapy by analyzing its levels in rectal tumors of patients with different responses to the therapy. Cellular mechanisms of FGFR4-induced radioresistance were analyzed by silencing or over-expressing FGFR4 in CRC cell line models. Our findings showed that the FGFR4 staining score was significantly higher in pre-treatment biopsies of non-responsive than responsive patients. Similarly, high expression of FGFR4 inhibited radiation response in cell line models. Silencing or inhibition of FGFR4 resulted in a reduction of RAD51 levels and decreased survival in radioresistant HT29 cells. Increased RAD51 expression rescued cells in the siFGFR4-group. In radiosensitive SW480 and DLD1 cells, enforced expression of FGFR4 stabilized RAD51 protein levels resulting in enhanced clearance of γ-H2AX foci and increased cell survival in the mismatch repair (MMR)-proficient SW480 cells. MMR-deficient DLD1 cells are defective in homologous recombination repair and no FGFR4-induced radioresistance was observed. Based on our results, FGFR4 may serve as a predictive marker to select CRC patients with MMR-proficient tumors who may benefit from pre-operative radiotherapy.

// Abigail J. Deloria 1 , Doris Hoflmayer 2, 6 , Philip Kienzl 1 , Justyna Łopatecka 1 , Sandra Sampl 1 , Martin Klimpfinger 2 , Tamara Braunschmid 3 , Fabienne Bastian 3 , Lingeng Lu 4 , Brigitte Marian 1 , Stefan Stattner 3, 5 , Klaus Holzmann 1 1 Division of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University Vienna, Austria 2 Department of Pathology and Bacteriology, Social Medical Center South, Kaiser Franz Josef Hospital, Vienna, Austria 3 Department of Surgery, Social Medical Center South, Kaiser Franz Josef Hospital, Vienna, Austria 4 Department of Chronic Disease Epidemiology, Yale School of Public Health, School of Medicine, Yale Cancer Center, Yale University, New Haven, USA 5 Department of Visceral, Transplantation and Thoracic Surgery, Innsbruck, Austria 6 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to: Klaus Holzmann, email: klaus.holzmann@meduniwien.ac.at Keywords: colorectal cancer, epithelial splicing regulatory protein, microsatellite instability, overall survival, prognostic marker Received: April 25, 2016 Accepted: September 02, 2016 Published: September 16, 2016 ABSTRACT ESRPs are master splice regulators implicated in alternative mRNA splicing programs important for epithelial-mesenchymal transition (EMT) and tumor progression. ESRP1 was identified in some tumors as good or worse predictor of outcome, but in colorectal cancer (CRC) the prognostic value of ESRPs and relation with mesenchymal splice variants is not clear. Here, we studied 68 CRC cases, compared tissue expression of ESRPs with clinical data and with EMT gene splice patterns of conditional CRC cells with deficient ESRP1 expression. Around 72% of patients showed global decreased transcript expression of both ESRPs in tumor as compared to matched non-neoplastic colorectal epithelium. Reduction of ESRP1 in tumor cells was evaluated by immunohistochemistry, associated with microsatellite stability and switch to mesenchymal splice signatures of FGFRs, CD44, ENAH and CTNND1(p120-catenin). Expression of ESRPs was significantly associated with favorable overall survival (log-rank test, P =0.0186 and 0.0408), better than prognostic stratification by tumor staging; and for ESRP1 confirmed with second TCGA cohort (log-rank test, P =0.0435). Prognostic value is independent of the pathological stage and microsatellite instability (ESRP1: HR=0.36, 95%CI 0.15–0.91, P =0.032; ESRP2: HR=0.23, 95%CI 0.08–0.65, P =0.006). Our study supports the role of ESRP1 as tumor suppressor and strongly suggests that ESRPs are candidate markers for early detection, diagnosis, and prognosis of CRC.

NSUN2 is a tRNA methyltransferase and plays an important role in cell development via modifying RNA methylation. We aimed to evaluate the expression of NSUN2 and its prognostic value in HNSCC. Random-effects model of meta-analysis shows 1.99-folds (95% CI: 1.89-2.09) upregulation of NSUN2 expression in HNSCC versus normal tissues. Patients with high NSUN2 levels had approximately 22 months shorter overall survival, and had a higher mortality risk than those with low one (p-trend = 0.020). In conclusion, NSUN2 is a potential independent prognostic marker and may be a potential therapeutic target in HNSCC.

Cannabidiol (CBD) and cannabidivarin (CBDV) are natural cannabinoids which are consumed in increasing amounts worldwide in cannabis extracts, as they prevent epilepsy, anxiety, and seizures. It was claimed that they may be useful in cancer therapy and have anti-inflammatory properties. Adverse long-term effects of these drugs (induction of cancer and infertility) which are related to damage of the genetic material have not been investigated. Therefore, we studied their DNA-damaging properties in human-derived cell lines under conditions which reflect the exposure of consumers. Both compounds induced DNA damage in single cell gel electrophoresis (SCGE) experiments in a human liver cell line (HepG2) and in buccal-derived cells (TR146) at low levels (≥ 0.2 µM). Results of micronucleus (MN) cytome assays showed that the damage leads to formation of MNi which reflect chromosomal aberrations and leads to nuclear buds and bridges which are a consequence of gene amplifications and dicentric chromosomes. Additional experiments indicate that these effects are caused by oxidative base damage and that liver enzymes (S9) increase the genotoxic activity of both compounds. Our findings show that low concentrations of CBD and CBDV cause damage of the genetic material in human-derived cells. Furthermore, earlier studies showed that they cause chromosomal aberrations and MN in bone marrow of mice. Fixation of damage of the DNA in the form of chromosomal damage is generally considered to be essential in the multistep process of malignancy, therefore the currently available data are indicative for potential carcinogenic properties of the cannabinoids. Electronic supplementary material The online version of this article (10.1007/s00204-018-2322-9) contains supplementary material, which is available to authorized users.

Aim of this study was to clarify if extension of the work phase has an impact on DNA- stability, telomere lengths and inflammatory markers. We conducted an intervention trial with office workers (n = 24) and carpenters (n = 10), who changed their working schedule from 8 to 12 h per day over a period of 3 months. The work of both groups involved only moderate physical activity. We found no evidence for induction of double strand breaks (measured in γH2AX assays) and relative telomere lengths (relTL_36B4 and ALB) in lymphocytes in the two study groups. Furthermore, no overall changes of the levels of C-reactive protein (CRP), interleukin-6 (IL-6) and thiobarbituric acid reactive substances (TBARS) in plasma were detected. However, we found in agreement with earlier investigations a moderate (not significant) increase of the CRP levels with age. Furthermore, significant higher CRP concentrations (P = 0.03) were detected in young individuals (21–30 years) as a consequence of the extended working period. Taken together our findings indicate that prolongation of the working hours has no pronounced impact on DNA stability, telomere shortening and inflammatory markers; but the increase of the CRP concentrations in young workers may be indicative for adverse health effects in this subgroup.

Malignant pleural mesothelioma is an aggressive malignancy characterized by frequent resistance to chemo- and radiotherapy, poor outcome, and limited therapeutic options. Fibroblast growth factors (FGFs) and their receptors are potential targets for cancer therapy, but their significance in mesothelioma has remained largely undefined.To investigate the antimesothelioma potential of FGF receptor 1 (FGFR1) inhibition.Expression of FGFs and their receptors was analyzed in mesothelioma cell lines and tissue specimens. Several cell models were used to investigate FGFR1 inhibition in vitro and in combination with cisplatin and irradiation. Mouse intraperitoneal xenotransplant models were used for in vivo validation.FGFR1, FGF2, and FGF18 were overexpressed in mesothelioma. Stimulation with FGF2 led to increased cell proliferation, migration, and transition to a more sarcomatoid phenotype in subsets of mesothelioma cell lines. In contrast, inhibition of FGFR1 by a specific kinase inhibitor or a dominant-negative FGFR1 construct led to significantly decreased proliferation, clonogenicity, migration, spheroid formation, and G1 cell cycle arrest in several mesothelioma cell lines, accompanied by apoptosis induction and decreased mitogen-activated protein kinase pathway activity. Reduced tumor growth, proliferation, mitogenic signaling, and apoptosis induction were observed in vivo. Inhibition of FGFR1 synergistically enhanced the cytotoxic effects of ionizing radiation and cisplatin.Our data suggest that the malignant phenotype of mesothelioma cells depends on intact FGF signals, which should be considered as therapeutic targets with a promising chemo- and radiosensitizing potential.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism (TMM) found in some human tumors such as sarcomas. Canine tumors are not characterized for ALT and the study aim was to identify if the ALT phenotype exists in canine sarcomas. Sixty-four canine sarcoma samples (20 snap-frozen, 44 FFPE) as well as six canine sarcoma cell lines were screened for ALT by C-circle assay. ALT was further evaluated by measuring telomere length via qPCR and telomere restriction-fragments including pulsed-field electrophoresis. ALT-associated proteins were validated by immunohistochemistry. Further, telomerase activity (TA) and gene expression were analyzed by TRAP and qPCR. DNA from 20 human neuroblastomas and 8 sarcoma cell lines served as comparative controls. ALT was detected in 9.4% (6/64) canine sarcomas including aggressive subtypes as hemangiosarcoma, osteosarcoma, and histiocytic sarcoma. C-circle levels were comparable with human ALT-positive controls. All ALT tumors demonstrated loss of ATRX expression and 5/6 showed strong p53 expression. TA was detected in 93% (14/15) snap-frozen samples including a sarcoma with ALT activity. This tumor showed long heterogeneous telomeres, and a high level of colocalization of DAXX with telomeres. One sarcoma was ALT and TA negative. All canine and human sarcoma cell lines were ALT negative. In this study, we demonstrated that canine sarcomas use ALT. As in humans, ALT was identified in aggressive sarcomas subtypes and coexisted with TA in one tumor. Overall, canine sarcomas seem to share many similarities with their human counterparts and appear an attractive model for comparative telomere research. © 2016 Wiley Periodicals, Inc.

To better understand the response of HCC to EGFR inhibition, we analyzed factors connected to the resistance of HCC cells against gefitinib. Sensitive HCC3 cells co-expressed EGFR and ErbB3 but lacked kinase-domain mutations in EGFR. Interestingly, expression of MVP was restricted to resistant cell lines, whereas ABCB1 and ABCC1 showed no association with gefitinib resistance. Moreover, ectopic MVP expression in HCC3 cells decreased gefitinib sensitivity, increased AKT phosphorylation and reduced the expression of inflammatory pathway-associated genes, whereas silencing of MVP in Hep3B and HepG2 cells increased sensitivity. These findings suggest MVP as a novel player in resistance against EGFR inhibition.

A hallmark of tumor cell survival is the maintenance of elongated telomeres. It is known that antiviral reverse transcriptase inhibitors (RTIs) such as azidothymidine (AZT) and didanosine (ddI) lead to telomere shortening at high, potentially toxic concentrations. We hypothesized that those drugs might have synergistic effects enabling successful therapy with low, nontoxic concentrations. Biologic effects of AZT and ddI were analyzed at concentrations that correspond to minimal plasma levels achieved during human immunodeficiency virus therapy. Long-term coapplication of low-dose AZT and ddI induced a significant shortening of telomeres in the tumor cell lines HCT-116, SkMel-28, MelJuso, and Jurkat. Treatment of cells with both RTI, but not with single RTI, led to a significant accumulation of γH2AX, to p53 phosphorylation, and to cell apoptosis in all cell lines. Oral low-dose dual RTI application but not low-dose single RTI application was associated with a significantly reduced tumor growth of HCT-116 cells in mice. This antiproliferative activity of the combined use of AZT and ddI at low, clinically applicable concentrations warrants clinical testing in human solid cancer.

Purpose: Melatonin (N-acetyl-5-methoxytryptamine) (MT) is a hormone that acts as an antioxidant. It is produced by the pineal gland and within the retina; its release is blocked by light entering the eye. We examined whether MT daytime levels differ between pseudophakic patients with age-related macular degeneration (ARMD) and pseudophakic subjects without any ocular pathology of the same age. Methods: A prospective, cross-sectional, observational study was performed. Pseudophakic patients of the same age group were included. Patients underwent complete ophthalmic examinations and blood sampling between 08:00 and 10:00 hr. MT daytime value in the serum was the main outcome measure. Results: Sixty-nine pseudophakic patients were included. Fifty patients with exudative and non-exudative ARMD were in the study group while 19 patients were controls. Patients with ARMD had significantly higher daytime levels of MT (P = 0.003). There were significant differences in MT daytime levels between the exudative and non-exudative forms (P = 0.009). MT values also correlated with the best-corrected visual acuity (r = )0.285, P = 0.019). Conclusion: These data indicate that pseudophakic patients with ARMD produce more MT during the day compared to pseudophakic subjects without ARMD. This may be caused by the reduced visual acuity in patients with ARMD, whereby less light reaches the photoreceptors, allowing MT secretion to continue during the day. Because MT also acts as an antioxidant and daytime levels are higher in patients with ARMD, these results might be interpreted as a rescue factor.

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.

Fibroblast growth factors (FGFs) and their high-affinity receptors contribute to the autocrine growth stimulation in several human malignancies. Here, we describe that FGF18 expression is up-regulated in 34/38 colorectal tumours and is progressively enhanced during colon carcinogenesis reaching very high levels in carcinoma. Moreover, our data suggest that FGF18 affects both tumour cells and tumour microenvironment in a pro-tumorigenic and pro-metastatic way. Addition of recombinant FGF18 to the culture media of slowly growing colorectal tumour cell lines LT97 and Caco-2 stimulated proliferation. Phosphorylation of externally regulated kinase 1/2 and S6 was increased already 5 min after growth factor addition. SW480 cells, endogenously producing large amounts of FGF18, were not affected in this setting, but recombinant FGF18 supported tumour cell survival under conditions of serum starvation. Down-modulation of endogenous FGF18 production by small interference RNA (siRNA) significantly reduced clonogenicity of SW480 cells and restored sensitivity to exogenous FGF18. With respect to the tumour microenvironment, both recombinant and tumour-derived FGF18 stimulated growth of colon-associated fibroblasts at 0.1 ng/ml and migration at 10 ng/ml. In addition, recombinant FGF18 (10 ng/ml) induced tube formation in human umbilical vein endothelial cells. siRNA knock down demonstrated that tube-forming activity of colon cancer cell supernatants depended to a large part on tumour cell-derived FGF18. In summary, this study demonstrates that FGF18 is almost generally over-expressed in colon cancer and exerts pro-tumorigenic effects both in the epithelial and the stromal compartments by stimulating growth and survival of tumour cells, migration of fibroblasts and neovascularization. Together, these data strongly support an oncogenic role of FGF18 in colorectal cancer.

Fibroblast growth factor receptors (FGFRs) are frequently up-regulated in subsets of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight that FGFR3 splice variants IIIb and IIIc impact considerably on the malignant phenotype of HCC cells. The occurrence of FGFR3 variants was analyzed in human HCC samples. In hepatoma/hepatocarcinoma cell lines, FGFR3 isoforms were overexpressed by lentiviral constructs or down-modulated by small interfering RNA (siRNA; affecting FGFR3-IIIb and -IIIc) or an adenoviral kinase-dead FGFR3-IIIc construct (kdFGFR3). Elevated levels of FGFR3-IIIb and/or -IIIc were found in 53% of HCC cases. FGFR3-IIIb overexpression occurred significantly more often in primary tumors of large (pT2-4) than of small size (pT1). Furthermore, one or both isoforms were enhanced mostly in cases with early tumor infiltration and/or recurrence at the time of surgery or follow-up examinations. In hepatoma/hepatocarcinoma cells, up-regulated FGFR3-IIIb conferred an enhanced capability for proliferation. Both FGFR3-IIIb and FGFR3-IIIc suppressed apoptotic activity, enhanced clonogenic growth, and induced disintegration of the blood/lymph endothelium. The tumorigenicity of cells in severe combined immunodeficiency mice was augmented to a larger degree by variant IIIb than by IIIc. Conversely, siRNA targeting FGFR3 and kdFGFR3 reduced clonogenicity, anchorage-independent growth, and disintegration of the blood/lymph endothelium in vitro. Furthermore, kdFGFR3 strongly attenuated tumor formation in vivo. Conclusions: Deregulated FGFR3 variants exhibit specific effects in the malignant progression of HCC cells. Accordingly, blockade of FGFR3-mediated signaling may be a promising therapeutic approach to antagonize growth and malignant behavior of HCC cells.(Hepatology 2015;62:1767–1778)

The major vault protein (MVP) is the main component of vaults, large ribonucleoprotein particles implicated in the regulation of cellular signaling cascades and multidrug resistance. Here, we identify MVP as an interferon gamma (IFN-gamma)-inducible protein. Treatment with IFN-gamma resulted in a significant upregulation of MVP promoter activity as well as mRNA and protein levels. Activation of MVP expression by IFN-gamma involved transcriptional upregulation through the JAK/STAT pathway based on an interaction of STAT1 with an interferon-gamma-activated site (GAS) within the proximal MVP promoter. Mutation of this site distinctly reduced basal as well as IFN-gamma-stimulated MVP transcription. IFN-gamma also significantly enhanced the translation rate of MVP. Ectopic MVP overexpression in the MVP-negative lung cancer cell model H65 led to a downregulation of three known IFN-gamma-regulated genes, namely ICAM-1, CD13 and CD36. Additionally, presence of MVP in H65 cells blocked both basal and IFN-gamma-induced ICAM-1 expression whereas downmodulation of endogenous MVP levels by shRNA enhanced IFN-gamma-induced ICAM-1 expression in U373 glioblastoma cells. MVP-mediated IFN-gamma insensitivity was accompanied by significantly reduced STAT1 phosphorylation at Y701 and diminished translocation of STAT1 into the nucleus. Summarizing, we identify MVP as an IFN-gamma-responsive gene interfering with IFN-gamma-activated JAK/STAT signals. These data further substantiate that the vault particle functions as a general interaction platform for cellular signaling cascades.

The major vault protein (MVP) has been implicated in multidrug resistance, cellular transport, and malignant transformation. In this study we aimed to identify crucial MVP promoter elements that regulate MVP expression. By mutation as well as deletion analysis a conserved proximal GC-box element was demonstrated to be essential for basal human MVP promoter transactivation. Binding of Sp-family transcription factors but not AP2 to this element in vitro and in vivo was shown by EMSA and ChIP assays, respectively. Inhibition of GC-box binding by a dominant-negative Sp1-variant and by mithramycin A distinctly attenuated MVP promoter activity. In Sp-null Drosophila cells, the silent human MVP promoter was transactivated by several human Sp-family members. In human cells the MVP promoter was potently stimulated by the histone deacetylase (HDAC) inhibitors butyrate (NaB) and trichostatin A (TSA), resulting in enhanced MVP expression. This stimulation was substantially decreased by mutation of the single GC-box and by application of mithramycin A. Treatment with HDAC inhibitors led to a distinct decrease of Sp1 but increase of Sp3 binding in vivo to the respective promoter sequence as demonstrated by ChIP assays. Summarising, this study identifies variations in Sp-transcription factor binding to a single proximal GC-box element as critical for basal MVP promoter activation and its stimulation by HDAC inhibitors.

Comparative genomic hybridization was used to map copy number abnormalities in 48 short-term cell cultures established from different stages and types of human melanoma. A variety of random and non-random chromosomal alterations were detected, with gains within chromosomes 20q, 7q, 7p, 20p, 6p and 17q and losses in 9p, 10q, 6q, 10p, 4q, and 11q being the most common observations. In addition, several other chromosomal loci were over- or under-represented in subgroups of melanomas. For example, sequences on 3q26 were over-represented in 33% and on 5p15.33 in 27% of cell cultures, reaching the level of amplification in 12% and 22%, respectively. These regions harbour the two essential genes for the enzyme telomerase: the telomerase reverse transcriptase gene (hTERT) on 5p15.33 and the telomerase RNA component gene (hTERC) on 3q26. Using fluorescence in situ hybridization and Southern blot analysis, both genes were shown to be over-represented or amplified in several melanomas. Interestingly, hTERT amplification was abundant in superficial spreading primary melanomas, subcutaneous metastases and malignant effusion-derived cells, but completely absent or very rare in primary nodular melanomas as well as brain, bone and lymph node metastases. Several chromosomes or chromosomal regions harbouring telomerase-suppressing activities (3p, 4, 6 and 10p) were frequently under-represented in melanomas. Our data suggest that genetic alterations at several chromosomal loci might facilitate activation of telomerase during the development of cutaneous malignant melanoma.

A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5′-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.

Amino acid sequencing and mass spectrometry revealed identity of a human nuclear matrix protein, termed hNMP 265, with a predicted protein of gene KIAA0111. Two-dimensional electrophoresis and Northern hybridization showed the protein to ubiquitously occur in various human cell types. Exhibiting DEAD-box motifs characteristic for RNA helicases, hNMP 265 is highly similar to the human initiation factors eIF4A-I and -II. On the other hand, hNMP 265 greatly differs from the initiation factors by a N-terminal sequence rich in charged amino acids. Sequence searches and alignments indicate proteins related to hNMP 265 in other eukaryotes. Chimeras between hNMP 265 and green fluorescence protein or hapten appeared as speckles in extranucleolar regions in the nucleus, but not in the cytoplasm. Experiments with tagged deletion mutants indicated that the N-terminal amino acid sequence is necessary for nuclear localization. A putative role of hNMP 265 in pre-mRNA processing is discussed.

To detect putative filament forming components, nuclear matrix proteins were searched for proteins extensively reassembling from urea solution. Eight proteins, ubiquitously occurring in various human cell types, but not apparent in the cytosol, were registered by means of two-dimensional gel electrophoresis. They consisted of a protein exhibiting a novel amino acid sequence; of nuclear lamin B2, RbAp46, and RbAp48; and of four as yet unknown proteins. Furthermore, partial sequencing, mass spectrometry, and immunodetection of proteins demonstrated the presence of molecular chaperones and protein folding catalysts in the nuclear matrix fractions. In addition to a TCP-1-related protein, certain members of the heat shock, PDI, and calreticulin family of proteins were detected. On the basis of the absence of several other heat shock proteins in the nuclear matrix fraction, a general contamination by cytoplasmic chaperones appears unlikely.

Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.

Fibroblast growth factor receptors (FGFRs) are important in malignant progression of several human epithelial tumors. However, little is known about FGFRs in canine or human soft tissue sarcomas. Thus, our aim was to investigate expression of FGFRs and their involvement in cell survival in sarcomas of both species. FGFR1-4 and FGFRL1 transcripts as well as IIIb/IIIc splice variants of FGFR1-3 were evaluated in 3 canine- and 6 human sarcoma cell lines and 19 spontaneous canine sarcomas by SYBRqPCR. FGFR1 protein expression was assessed by immunohistochemistry. Growth inhibitory effects of FGFR1 inhibitor PD166866 and dominant negative recombinant FGFR adenoviral expression constructs (dnFGFR) on tumor cell lines were analyzed. Profiling of multiple FGFR transcripts detected comparable co-expression in most of human and canine sarcoma cell lines and canine tumor specimens. This indicates existence of closely related regulation mechanisms for FGFR expression in sarcomas of both species. FGFR1 with splice variant IIIc was consistently expressed with highest transcript levels. In 88% of the spontaneous tumor samples a heterogeneous FGFR1 protein expression was observed. Significant growth inhibition and cell death was seen after infection with dnFGFR1 in canine and human sarcoma cells, but not with dnFGFR3 and 4. PD166866 showed selective cytotoxicity with IC50 values between 12.1 and 26.4 μM. FGFR1 inhibition blocked ligand-induced tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase isoforms. This study emphasizes the important role FGFR1, especially splice variant IIIc, likely plays in sarcomas. Inhibitory small molecules could be of potential use for targeted therapy in aggressive sarcomas of both species.

A gly388arg polymorphism (rs351855) in the transmembrane domain of the fibroblast growth factor receptor (FGFR4) is associated with increased risk, staging, and metastasis in several different types of cancer. To specifically assess the impact of the polymorphic FGFR4 in colorectal cancer (CRC), we engineered CRC cell lines with distinct endogenous expression patterns to overexpress either the FGFR4gly or FGFR4arg alleles. The biologic analyses revealed an oncogenic importance for both polymorphic alleles, but FGFR4gly was the stronger inducer of tumor growth, whereas FGFR4arg was the stronger inducer of migration. An evaluation of clinical specimens revealed that FGFR4 was upregulated in 20/71 patients independent of gly388arg status. There was no correlation between the presence of an FGFR4arg allele and CRC or polyp risk in 3,471 participants of the CORSA study. However, among 182 patients with CRC, FGFR4arg-carriers had a fivefold higher risk of tumors that were stage II or greater. Together, our results established that both allelic forms of FGFR4 exert an oncogenic impact and may serve equally well as therapeutic targets in CRC. One important implication of our findings is that FGFR4arg-carriers are at a higher risk for more aggressive tumors and therefore may profit from early detection measures. Cancer Res; 72(22); 5767–77. ©2012 AACR.

Fibroblast growth factors (FGFs) and their high-affinity receptors [fibroblast growth factor receptors (FGFRs)] contribute to autocrine and paracrine growth stimulation in several nonliver cancer entities. Here we report that at least one member of the FGF8 subfamily (FGF8, FGF17, and FGF18) was up-regulated in 59% of 34 human hepatocellular carcinoma (HCC) samples that we investigated. The levels of the corresponding receptors (FGFR2, FGFR3, and FGFR4) were also elevated in the great majority of the HCC cases. Overall, 82% of the HCC cases showed overexpression of at least one FGF and/or FGFR. The functional implications of the deregulated FGF/FGFR system were investigated by the simulation of an insufficient blood supply. When HCC-1.2, HepG2, or Hep3B cells were subjected to serum withdrawal or the hypoxia-mimetic drug deferoxamine mesylate, the expression of FGF8 subfamily members increased dramatically. In the serum-starved cells, the incidence of apoptosis was elevated, whereas the addition of FGF8, FGF17, or FGF18 impaired apoptosis, which was associated with phosphorylation of extracellular signal-regulated kinase 1/2 and ribosomal protein S6. In contrast, down-modulation of FGF18 by small interfering RNA (siRNA) significantly reduced the viability of the hepatocarcinoma cells. siRNA targeting FGF18 also impaired the cells' potential to form clones at a low cell density or in soft agar. With respect to the tumor microenvironment, FGF17 and FGF18 stimulated the growth of HCC-derived myofibroblasts, and FGF8, FGF17, and FGF18 induced the proliferation and tube formation of hepatic endothelial cells. Conclusion: FGF8, FGF17, and FGF18 are involved in autocrine and paracrine signaling in HCC and enhance the survival of tumor cells under stress conditions, malignant behavior, and neoangiogenesis. Thus, the FGF8 subfamily supports the development and progression of hepatocellular malignancy. (HEPATOLOGY 2011)

Background: Deregulation of fibroblast growth factor receptor 3 (FGFR3) is involved in several malignancies. Its role in colorectal cancer has not been assessed before. Methods: Expression of FGFR3 in human colorectal tumour specimens was analysed using splice variant-specific real-time reverse transcriptase PCR assays. To analyse the impact of FGFR3-IIIc expression on tumour cell biology, colon cancer cell models overexpressing wild-type (WT-3b and WT3c) or dominant-negative FGFR3 variants (KD3c and KD3b) were generated by either plasmid transfection or adenoviral transduction. Results: Although FGFR3 mRNA expression is downregulated in colorectal cancer, alterations mainly affected the FGFR3-IIIb splice variant, resulting in an increased IIIc/IIIb ratio predominantly in a subgroup of advanced tumours. Overexpression of WT3c increased proliferation, survival and colony formation in all colon cancer cell models tested, whereas WT3b had little activity. In addition, it conferred sensitivity to autocrine FGF18-mediated growth and migration signals in SW480 cells with low endogenous FGFR3-IIIc expression. Disruption of FGFR3-IIIc-dependent signalling by dominant-negative FGFR3-IIIc or small interfering RNA-mediated FGFR3-IIIc knockdown resulted in inhibition of cell growth and induction of apoptosis, which could not be observed when FGFR3-IIIb was blocked. In addition, KD3c expression blocked colony formation and migration and distinctly attenuated tumour growth in SCID mouse xenograft models. Conclusion: Our data show that FGFR3-IIIc exerts oncogenic functions by mediating FGF18 effects in colorectal cancer and may constitute a promising new target for therapeutic interventions.

Expression of transgenes from adenovirus vectors has become an extremely important and widely used tool in experimental cancer research and many other areas in the life sciences. It needs to be kept in mind, however, that adenoviruses are human pathogens and avoiding exposure of laboratory personnel to infectious viral particles is therefore an important concern. This issue seems even more important when the transgenes expressed for experimental purposes include oncogenic sequences. Decontamination procedures are thus required, whenever laboratory experiments with adenovirus vectors are performed and the effectiveness of these procedures has to be established. While many reports exist on the decontamination of blood and pharmaceutical products, data on the stability of adenoviruses during experiments performed in most life science laboratories are very scarce. One reason for this is that many of the methods used for assessing viral decontamination are time consuming and laborious and cannot easily be incorporated into the broad range of experimental setups typically performed in the laboratory. In this chapter we describe a reliable, sensitive, and simple method for the assessment of adenovirus decontamination by the use of an adenovirus expressing green fluorescent protein (GFP). The GFP adenovirus is subjected to various test conditions and afterwards susceptible indicator cells are exposed to the recovered virions. GFP expression is detected by a combination of fluorescence microscopy and flow cytometry. The simplicity and flexibility of the method allows one to monitor viral decontamination during the different scenarios occurring in the life science laboratory.

Fibroblast growth factors (FGF) and their high-affinity receptors (FGFR) represent an extensive cellular growth and survival system. Aim of this study was to evaluate the contribution of FGF/FGFR-mediated signals to the malignant growth of non-small cell lung cancer (NSCLC) and to assess their potential as targets for therapeutic interventions. Multiple FGFR mRNA splice variants were coexpressed in NSCLC cells (n = 16) with predominance of FGFR1. Accordingly, both expression of a dominant-negative FGFR1 (dnFGFR1) IIIc-green fluorescent protein fusion protein and application of FGFR small-molecule inhibitors (SU5402 and PD166866) significantly reduced growth, survival, clonogenicity, and migratory potential of the majority of NSCLC cell lines. Moreover, dnFGFR1 expression completely blocked or at least significantly attenuated s.c. tumor formation of NSCLC cells in severe combined immunodeficient mice. Xenograft tumors expressing dnFGFR1 exhibited significantly reduced size and mitosis rate, enhanced cell death, and decreased tissue invasion. When FGFR inhibitors were combined with chemotherapy, antagonistic to synergistic in vitro anticancer activities were obtained depending on the application schedule. In contrast, simultaneous blockage of FGFR- and epidermal growth factor receptor-mediated signals exerted synergistic effects. In summary, FGFR-mediated signals in cooperation with those transmitted by epidermal growth factor receptor are involved in growth and survival of human NSCLC cells and should be considered as targets for combined therapeutic approaches. [Mol Cancer Ther 2008;7(10):3408–19]

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 down-modulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.

Upregulation of cyclooxygenase-2 (COX-2) and prostaglandin-dependent vascularisation in small adenomatous polyps is an essential part of colon carcinogenesis. To study the underlying cellular mechanisms, LT97 and Caco2 human colorectal tumour cells not expressing endogenous COX-2 were exposed to 1 microM prostaglandin E(2) (PGE(2)) in their medium. At 30 min after addition, expression of c-fos was stimulated 5-fold and 1.3-fold, respectively, depending on the activation of both extracellular signal-regulated kinase and p38. The amount of c-jun in nuclear extracts was increased 20% in LT97 cells. Expression of COX-2 was upregulated 1.7-fold in LT97 cells and 1.5-fold in Caco2 2 h after prostaglandin (PG) addition by a p38-mediated pathway. The known PGE(2) target gene vascular endothelial growth factor (VEGF) was not modulated. Effects of sustained PGE(2) production were studied in VACO235 cells that have high endogenous COX-2 and in LT97 cells infected with an adenovirus expressing COX-2. Prostaglandin E(2) secretion into the medium was 1-2 nM and 250 pM, respectively. Expression of both VEGF and c-fos was high in VACO235 cells. In LT97 cells, COX-2 upregulated c-fos expression and c-jun content in nuclear extracts 1.7- and 1.2-fold, respectively, in a PG-dependent way. This shows that exogenous PGE(2) as well as COX-2 overexpression affect signalling and gene expression in a way that enhances tumour progression.

12(S)-Lipoxygenase (LOX) is regarded as a pro-tumorigenic enzyme and as a potential target for therapy and prevention of cancer so that the search for specific 12(S)-LOX inhibitors is part of drug development strategies. To facilitate the identification of specific 12(S)-LOX inhibitors we have created an assay cell line by introducing a12(S)-LOX expression vector into SW480 colorectal cancer cells. When arachidonic acid was supplied in the medium both transiently and stably overexpressing cells produced 12(S)-hydroxytetraenic acid (HETE) originating from the transfected gene at 4-5-fold the amount obtained from control transfectants. 12(S)-HETE production was 1913.7+/-17.2pg/ml and reached a steady state level 24h after addition of arachidonic acid. To demonstrate the models suitability of 12(S)-LOX overexpressing SW480 cells they were used to measure the inhibitory activity of the plant phenols baicalein, kaempferol, quercetin, nordihydroguaretic acid and resveratrol which are known for their chemopreventive as well as LOX-inhibitory activity in different tumour models. All 5 compounds inhibited 12(S)-HETE production at concentrations below those necessary for growth inhibition.

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources.

Transcripts of genes encoding proteins of clathrin complexes have been reported to undergo tissue-specific alternative splicing. AP17, encoded by human CLAPS2 cDNA, is the small chain of the major clathrin adaptor complex AP-2 associated with mammalian plasma membranes. In this study, two cDNAs were isolated from a cDNA library of human blood cells. Whereas one cDNA encoded AP17, the other cDNA encoded a putative novel protein variant, termed AP17Delta. Both coding regions were completely sequenced. Consisting of 142aa residues, the predicted protein AP17Delta of 12kDa lacks 38aa residues of AP17. Using specific primers for RT-PCR, mRNAs for AP17Delta and AP17 were found in leukocytes and cultured leukemia cells. The finding of a putative intron in a human EST cDNA clone suggests that mRNAs for AP17 and AP17Delta are formed by alternative splicing. In addition, the identity of human and rat AP17 amino acid sequences is demonstrated.

Survivin protein accomplishes two basic functions: cell cycle regulation and control of apoptosis. It is only expressed in G2/M phase and it influences rescue pathways in apoptosis-induced cells. Overexpression of constitutive active c-H-ras in HeLa, or induction of c-H-ras in a stable HeLaDiR cell line, led to sustained survivin expression in all cell cycle phases and even protected cells from drug induced apoptosis. siRNA-mediated silencing of survivin reversed this protection. Here we link the anti-apoptotic property of survivin to its cell cycle (in)dependent regulation via the activity of oncogenic c-H-ras.

Cutaneous melanoma is a tumor with rising incidence and a very poor prognosis at the disseminated stage. Melanomas are characterized by frequent mutations in BRAF and also by overexpression of fibroblast growth factor 2 (FGF2), offering opportunities for therapeutic intervention. We investigated inhibition of FGF signaling and its combination with dacarbazine or BRAF inhibitors as an antitumor strategy in melanoma. The majority of melanoma cell lines displayed overexpression of FGF2 but also FGF5 and FGF18 together with different isoforms of FGF receptors (FGFRs) 1-4. Blockade of FGF signals with dominant-negative receptor constructs (dnFGFR1, 3, or 4) or small-molecule inhibitors (SU5402 and PD166866) reduced melanoma cell proliferation, colony formation, as well as anchorage-independent growth, and increased apoptosis. DnFGFR constructs also significantly inhibited tumor growth in vivo. Combination of FGF inhibitors with dacarbazine showed additive or antagonistic effects, whereas synergistic drug interaction was observed when combining FGFR inhibition with the multikinase/BRAF inhibitor sorafenib or the V600E mutant-specific BRAF inhibitor RG7204. In conclusion, FGFR inhibition has antitumor effects against melanoma cells in vitro and in vivo. Combination with BRAF inhibition offers a potential for synergistic antimelanoma effects and represents a promising therapeutic strategy against advanced melanoma.

SummaryCancer-related fibrin deposition and fibrinolysis were investigated by two-dimensional gel electrophoresis of human solid tumor and effusion specimen in addition to plasma samples. Fibrinogen gamma-chain dimer indicating fibrin deposition and plasmin-generated fibrinogen beta-chain fragments were identified in various solid tumor types by amino acid sequencing, mass spectrometry analysis and Western blotting. In tumor-associated effusions, these techniques allowed to observe plasmin-generated fragments of fibrinogen alpha, beta and gamma-chains in addition to elevated levels of acute-phase proteins. Similar observations were made in case of inflammation-associated effusions. No fibrin degradation product was observed in plasma samples, however, high amounts of fibrinogen gamma-chain dimer crosslinked by transglutaminase were detected in plasma from tumor patients, but not in plasma from controls and patients suffering acute infections and/or inflammations. This finding demonstrated that high transglutaminase activity may be associated with cancer. The presented data indicate that the amount of crosslinked fibrinogen gamma-chain dimer in plasma may correlate with tumor-associated fibrin deposition. The tumor-biological relevance of this potential marker protein is discussed.

Telomerase reactivation and expression of human telomerase gene [human telomerase reverse transcriptase (hTERT)] are hallmarks of unlimited proliferation potential of cancer cells. A polymorphic tandem repeats minisatellite of hTERT gene, termed MNS16A was reported to influence hTERT expression. To assess the role of MNS16A as potential biomarker for colorectal cancer (CRC), we investigated for the first time the association of MNS16A genotypes with risk of colorectal polyps and CRC. In the ongoing colorectal cancer study of Austria (CORSA), 3842 Caucasian participants were recruited within a large screening project in the province Burgenland including 90 CRC cases, 308 high-risk polyps, 1022 low-risk polyps and 1822 polyp free controls verified by colonoscopy. MNS16A genotypes were determined by polymerase chain reaction from genomic DNA. Associations of MNS16A genotypes with CRC risk were estimated by logistic regression analysis computing odds ratios (ORs) and 95% confidence intervals (CIs). We identified five different variable number of tandem repeats (VNTRs) of MNS16A including VNTR-364, a newly discovered rare variant. VNTR-274 allele was associated with a 2.7-fold significantly increased risk of CRC compared with the VNTR-302 wild-type (OR = 2.69; 95% CI = 1.11–6.50; P = 0.028). In our CORSA study, the medium length VNTR-274 was identified as risk factor for CRC. Although, this population-based study herewith reports the largest cohort size concerning MNS16A thus far, further large-scale studies in diverse populations are warranted to confirm hTERT MNS16A genotype as potential biomarker for assessment of CRC risk.